High precision quantification of sub-visible particles

ABSTRACT

The method is for quantification of sub-visible particles. A filter membrane is provided that has a plurality of pores defined therethrough. The filter membrane is in operational engagement with a vacuum chamber. The pores are sealed with a sealant. A sample droplet, containing a liquid and sub-visible particles, is applied onto the filter membrane. The liquid dissolves the sealant in pores disposed directly below the sample droplet. The liquid flows through the pores in which the sealant has been dissolved and the sub-visible particles remain on top of the filter membrane. The particles are enumerated in an electron microscope.

PRIOR APPLICATIONS

This is a continuation application that claims priority from U.S. patentapplication Ser. No. 15/736,621, filed on 14 Dec. 2017 that claimspriority from PCT patent application no. PCT/US2016/058011, filed on 21Oct. 2016 that claims priority from U.S. provisional patent applicationNo. 62/269,465, filed on 18 Dec. 2015.

TECHNICAL FIELD

The present invention relates to a method for high precisionquantification of sub-visible particles, such as micro-particles and/ornanoparticles, using microscopy such as scanning electron microscopy(SEM).

BACKGROUND AND SUMMARY OF THE INVENTION

A precise enumeration of the number of sub-visible particles such asvirus particles, virus-like particles, inorganic and polymeric beads andother nanoparticles and micro-particles from liquid samples is importantin many processes. For example, modified virus vectors are commonly usedin gene therapy applications. The number of active vectors per mL (theinfectious titer of the virus sample) can be determined using standardinfectivity assays. However, by using the currently available methods,it is not possible to precisely determine the total number of particles,including non-infectious particles, in the sample. The ratio ofinfectious over non-infectious particles provides invaluable informationabout the quality and efficacy of the final gene therapy product and theupstream development processes.

One major limitation of the currently available techniques, such asquantitative flow cytometry (QFCM), is that the nanoparticles ofinterest are not directly detected. Instead, the number of bound probesto a population of nanoparticles is quantitated. Since the number ofprobes that binds per nanoparticle varies, the precision of theconventional indirect techniques is typically low and dependent on theaffinity between the specimen and probe. A technique where thenanoparticle of interest could be directly detected would overcome thislimitation.

Moreover, if the technique would be able to visualize the particles atsufficient resolution, individual particles could be identified based ontheir size and morphology and thus be directly enumerated. Evenparticles within clusters could be enumerated and estimated. This is notpossible by using the currently available affinity methods or lightscattering-based techniques.

The novel high-precision direct particle method of the present inventionmay be used to enumerate both inorganic and organic sub-visibleparticles, such as nanoparticles, from liquid samples. One importantfeature is that the specimens are applied on a well-defined andmeasurable footprint. Another important feature is that the specimensare more evenly distributed than what has been possible before and thisreduces the need for sampling and it is now possible to conduct theanalysis at a resolution where the individual particles can easily beidentified. The sub-visible particles are directly detected without theneed for signal probes and can be visualized in normal two-dimensionalimages. The particle quantification SEM (pqSEM) method of the presentinvention is preferably based on low-vacuum filtering, scanning electronmicroscopy (SEM) or other electron microscopy techniques and imageanalysis. The present invention can be used with or without internalstandards, of which an example would be National Institute of Standardsand Technology (NIST) characterized polystyrene beads.

The present invention provides a solution to the above describedproblems. More particularly, the method is for quantification ofsub-visible particles. A filter membrane is provided that has aplurality of pores defined therethrough. The filter membrane is inoperational engagement with a vacuum chamber. The pores are sealed witha sealant. A sample droplet, containing a liquid with sub-visibleparticles, is applied onto the filter membrane. The liquid dissolves thesealant in the pores located directly below the sample droplet. Theliquid flows through the pores in which the sealant has been dissolvedand the sub-visible particles remain on top of the filter membrane. Thefilter membrane, with the particles disposed thereon, is moved to anelectron microscope and enumerated in images acquired in the microscope.

The method further comprises the step of pre-mounting a filter assembly,containing the filter membrane, onto a SEM support.

The method further comprises the step of placing a mounting tape on theSEM support.

The method further comprises the step of providing the SEM support,having an elongate channel defined therein, using an injector containingthe sample droplet, and aligning the injector on top of an elongatechannel prior to depositing the sample droplet on the filter membrane.

The method further comprises the step of connecting the SEM support to avacuum chamber connected to a vacuum source and subjecting the filtermembrane to a suction force via the elongate channel.

The method further comprises the step of depositing the sample dropletonto the filter membrane without the sample droplet touching any outsideedge of the filter membrane.

The method further comprises the step of the liquid only dissolving thesealant in the pores disposed directly below the sample droplet whilethe adjacent pores on the side of the droplet remain sealed with thesealant because the liquid has not been in contact with the sealantdisposed in those pores.

The method further comprises the step of the sub-visible particlesforming a defined and measurable footprint on the filter membrane andacquiring a series of images of the particles from an outside peripheryof the footprint to the center of the footprint.

The method further comprises the step of counting the particles in theelectron microscopy images acquired at a resolution where the particlesare clearly visible—either manually or automatically using imageanalysis methods.

The method further comprises the step of estimating the total area ofthe footprint on the filter membrane in microscopy images covering thewhole footprint (either one low-magnification image covering the wholefootprint or several higher magnification sub-images of the footprintstitched together).

The method further comprises the step of calculating the total number ofparticles in the sample from the area of the whole footprint and thenumber of particles per area unit derived from images at a resolutionhigh enough to clearly see single particles.

The method further comprises the step of possibly compensating foruneven radial particle distribution of the particles in the footprintfor which information is derived from acquiring a series of images fromthe periphery of the footprint through the center at a high enoughmagnification to clearly see individual particles.

The method further comprises the step of calculating the concentrationof particles in the solution using the total particle estimate from thefootprint; the applied volume and dilution of the liquid sample.

The method further comprises the step of using a diluent of the liquidto dissolve the sealant in the pores located directly below the sampledroplet. The specimen should be in a liquid form and the diluent shouldbe compatible with the diluent and have the property of effectivelydissolving the sealant that is being used.

The method further comprises the step of using glycine as the sealant.Other sealants that could be used include, but are not limited to,trehalose/sucrose-based sealants.

BRIEF DESCRIPTION OF DRAWINGS

FIG. 1 is a schematic elevational side view of a vacuum device of thepresent invention;

FIG. 2 is a schematic cross-sectional side view of the filter assembly;

FIG. 3A is an unprocessed high magnification SEM image of polystyrenebeads adhered to a poly-ether sulfone filter;

FIG. 3B is a detected and enumerated high magnification SEM image ofpolystyrene beads adhered to a poly-ether sulfone filter;

FIG. 3C is a close-up view of the SEM image of FIG. 3B above;

FIG. 4A is a schematic view of an edge of the droplet footprint imageusing SEM (primary electrons) at high magnification;

FIG. 4B is schematic view of an edge of the droplet footprint imageusing SEM (primary electrons) at low magnification;

FIG. 5A is a cross-sectional schematic side view of the membrane of thepresent invention with open pores;

FIG. 5B is a cross-sectional schematic side view of the membrane shownin FIG. 5A but with sealed pores;

FIG. 5C is a cross-sectional schematic side view of the membrane of thepresent invention shown in FIG. 5B having a sample droplet depositedthereon;

FIG. 5D is a cross-sectional schematic side view of the membrane shownin FIG. 5C having the droplet being absorbed into the membrane of thepresent invention; and

FIG. 6 is an enlarged view of FIG. 5D.

DETAILED DESCRIPTION

The method of the present invention is described with reference to FIGS.1-6. FIG. 1 is a schematic front view of a vacuum assembly 100 that hasa vacuum device 102 connected to a vacuum chamber 104 via a tubing 106extending therebetween. Preferably, the tubing 106 has a suitable valvesuch as a luer valve 108. A vacuum manometer 110 is in operativeengagement with the vacuum chamber 104 to measure a vacuum pressuretherein. A filter assembly 112 is mounted by a filter assembly mount 114on top of the vacuum chamber 104. A filter membrane 116 is disposed onthe filter assembly 112. An injector 118 is located above the filtermembrane 116.

FIG. 2 is a schematic cross-sectional view of the filter assembly 112.The entire analysis process of the present invention may be simplifiedby pre-mounting the filter membrane 116 onto the SEM support (aluminastub) 120 instead of doing it manually when the filter membrane 116contains the sample/specimen to be analyzed and enumerated. The mountingmay be done by simply drilling a hole in the SEM stub 120. The use ofsuch a device minimizes the risk of losing specimen or damaging thefilter membrane 116 during the previous setup in which the filtermembrane 116 that contains the specimen is handled manually during themounting onto the SEM stub 120. The details of the preparation of thefilter membrane 116 are discussed below. Such a pqSEM analyticalconsumable device is relatively inexpensive to manufacture.

More particularly, the filter assembly 112 preferably has a modified SEMalumina stub 120 onto which a double-sided carbon mounting tape 122 isplaced. The sealed porous filter membrane 116 is placed on top of thecarbon mounting tape 122. The process of sealing the filter membrane 116is described in detail below particularly with reference to FIGS. 5-6.The injector 118, that contains a specimen or sample 124 to be analyzed,is disposed or positioned above the filter membrane 116 and is used todeposit a sample droplet 126 onto the filter membrane 116. Because thestub 120 is sealingly connected to the filter assembly mount 114 that,in turn, is mounted on the vacuum chamber 104, there is vacuum insidethe stub 120 so that the vacuum exerts a suction force on the filtermembrane 116 from below the filter assembly 112. This is enabled becausean elongated cavity or channel 128, defined inside the stub 120, is influid communication with the filter membrane 116 and the vacuum chamber104. As described below, it is important that the injector 118 iscorrectly positioned above the filter membrane 116 so that when thesample droplet 126 is deposited onto the filter membrane 116, the sampledroplet 126 is not in contact with edges of the filter membrane 116 andplaced directly above an enlarged cavity portion 129 that is definedbetween channel 128 and the underside of the mounting tape 122.Preferably, the droplet 126 is placed at or near the center of thecavity portion 129 that is aligned with a longitudinal axis (L) thatextends through the channel 128.

FIGS. 3A-3C are high magnification SEM images of polystyrene beadsadhered to a poly-ether sulfone filter. FIG. 3A is an unprocessed image130 and FIG. 3B is a detected and enumerated image 132. FIG. 3C is aclose-up view 133 of the view of FIG. 3B and section 131 of FIG. 3A. Inthese example images, 77 particles were detected in a field of view of131.47 μm². This corresponds to approximately 0.59 particles per μm².View 133 shows the numbered particles 1, 2, 3, 4, 5, 6, 7, 28, 29, 30.

FIGS. 4A-4B show the edge of the droplet footprint imaged using SEM(primary electrons). FIG. 4A show an image 134 at high magnification andFIG. 4B shows a footprint image 136 at low magnification. The locationof the high magnification image 134, shown in FIG. 4A, is marked with awhite arrow in FIG. 4B so that image 134 shows a portion of the entirefootpring image 136. The edge of the droplet is well-defined with anegligable number of particles outside the footprint. At lowmagnification, the entire footprint 136 of the droplet is visualized andthe area of the footprint can be precisely measured. In this example,the area of the footprint (A_(total)) was measured to 8.436 mm².

FIGS. 5A-5D are cross-sectional side views of the filter membrane 116and describe the process of sealing the filter membrane 116 and thendissolving the sealant. In FIG. 5A, the filter membrane 116 has openpores 138, defined between elongate grid members 139 of the filtermembrane 116, that extends through the filter membrane 116. In FIG. 5B,the pores 138 are filled with a sealant 140. In FIG. 5C, the sampledroplet 126 of sample 124, that is a liquid 144 containing particles 142to be analyzed, is deposited onto the filter membrane 116. Preferably,the droplet 126 is deposited onto the filter membrane 116 by using theinjector 118 described above. Upon contact by droplet 126 with sealant140, liquid 144 dissolves sealant 140 that is disposed immediately belowdroplet 126 so that the liquid 144 is absorbed and passed through thepores 138 only disposed below droplet 126. Because particles to beenumarated 142 have a size that is greater than pores 138 of themembrane, the particles 142 are deposited on top the filter membrane 116while the liquid and any smaller contaminants 144 are absorbed or flowsinto the pores 138 below the droplet 126 as the sealant 140 in thosepores are dissolved and the liquid 144 is subject to the suction fromelongate chamber 128 of vacuum chamber 104 below filter membrane 116.

FIG. 6 is an enlarged view of the filter membrane 116 of FIG. 5D. Thesub-visible particles 142 rest on the filter membrane 116 and theparticles 142 are larger than the pores 138 a-138 j so they do not passthrough the pores even if the pores are open and subject to suction fromthe vacuum chamber 104 (shown in FIG. 1). Pores 138 a-d and 138 i-j arestill filled with sealant 140 since they have not been dissolved by theliquid 144 because they have not been in contact with the liquid 144 ofsample droplet 126 (see FIG. 5C). More particularly, the diluent, suchas a suitable buffer, in liquid 144 dissolves sealant 140. As indicatedabove, the diluent should be compatible with the specimen i.e. haveminimal influence of the specimen morphology and aggregation state. Thediluent should also have the property of dissolving the sealant. This isto make sure that the vacuum is only maintained at the footprint and sothat the setup does not lose vacuum by “opening up” pores outside thearea of interest. As the liquid 144 dissolves the sealant 140 disposedin pores 138 e-138 h, the liquid 144 fills pores 138 e-138 h to replacethe sealant 140. This makes it easier to enumerate particles 142 becauseparticles 142 are laying on top of filter segment 116 and arewell-distributed across the filter membrane 116.

EXAMPLE

Below is an illustrative example of method of preparing the filtermembrane 116 according to the present invention.

1. A sample, containing sub-visible particles 142, such asmicro-particles and/or nanoparticles, is prepared for enumeration bydiluting the sample in series in an appropriate diluent (typicallywater, phosphate-buffered, HEPES-buffered, TRIS-buffered orHistidine-buffered saline) depending on the buffer conditions of eachparticular sample.

2. A fixation agent (typically glutaraldehyde or formaldehyde) and/or astabilizing agent (typically sucrose or glycerol) can be introduced intothe diluted sample solution 124, that also includes the sub-visibleparticles 142, to stabilize and preserve the structure of the particlesand in some samples prevent undesirable aggregation of the particles142. The fixation/stabilizing agents and the diluent correspond toliquid 144 and together with the particles 142 form the sample/specimen124 and sample droplet 126. The fixation/stabilizing agents are used toprevent the particles 142 from being destroyed or damaged duringhandling and from undesirably adhering to one another which make it moredifficult to later enumerate the particles 142.

3. The filter assembly 112 consists of the porous filter membrane 116(typically made of poly-ether sulfone or polycarbonate) with pores 138that have a defined pore size (typically 0 to 15 nm) and an openablefilter cassette made of plastic or equivalent are used for separatingthe particles 142 from the liquid. A suitable filter assembly 112 isbest shown in FIG. 2. In general, the filters are bought in bulk assingle-use filters and thus need to be mounted on something. Somevendors also sell filter holders and these devices are originally madeto be connected to a syringe and push the liquid through and thus notsucking the liquid through using vacuum. It is therefore necessary tomount the filter to a filter assembly to assure vacuum integrity. Aftersome experimentation it was surprisingly realized that the filter couldbe mounted directly on the SEM support which saves time and avoids thecritical steps of manually handling the specimen containing filters. Itis conceivable that such an assembly can be inexpensively made and besold as a SEM consumable.

4. The filter assembly 112 is mounted onto the top of the plastic vacuumchamber 104 which in turn is connected to the vacuum device 102 viatubing 106.

5. The vacuum in the vacuum chamber 104 is controlled by the 3-way Luervalve 108 and monitored by using the vacuum manometer 110. An automaticsystem using magnetic valves controlled by an electronic monitoringsystem can also be implemented.

6. The pores 138 in the filter membrane 116 are preferably sealed withsealant 140 such as glycine (or equivalent) prior to sample applicationof the sample droplet 126, as best shown in FIGS. 5B and 5C. It wassurprisingly and unexpectedly discovered that by using sealant 140 inthe filter membrane 116, the particles 142 inside droplet 126 weredistributed more evenly (prior to removing the liquid 144 of the droplet126) and there was no need to use a high vacuum force to reduce the riskof the droplet spreading out unevenly on the filter membrane. It shouldbe noted that the distribution of the particles does not have to be thesame at the outer periphery and as it is at the center. The pattern ofthe particle distribution can be determined by scanning the footprint134/136 (see FIGS. 4A-4B) of the particle sample, disposed on the filtermembrane, from the outer periphery or outer edge 137 of the foot print136 towards the center 139 of the footprint of the particle sample. Ifthe scanned portion of the particle sample shows a certain pattern ofdistribution of particles, it can be reliably assumed that the sameparticle distribution pattern apply around the entire circular-shapedparticle sample footprint 136 partly because the particles were giventime to settle before the sealant 140 is dissolved by the liquid in thedroplet 126. Because the droplet 126 is first deposited onto the sealedfilter membrane 116 the outer edge 137 of footprint 136 of the droplet126 becomes relatively distinct or sharp which is important in order todetermine where to start the enumeration and scanning towards the center139 of the circular-shaped particle sample or footprint 136 deposited onthe dissolved filter membrane 116. It was unexpectedly discovered thatthe advantages of the relatively even distribution of the particles onthe filter membrane outweighed the drawbacks of having to remove thesealant to permit the liquid in the droplet to flow through the filtermembrane before starting the enumeration of the particles. Any uneven ornon-distinct periphery of the footprint of the droplet on the filtermembrane makes it more difficult to determine the footprint thereof andknow which area is to be analyzed in order to count all the particles inthe droplet. By applying the sample droplet 126 onto the filter membrane116, with all the pores 138 being sealed by sealant 140, the particles142 are evenly distributed inside droplet 126 as the droplet 126 spreadsout on the sealed top surface of filter membrane 116. The requirement ofhaving to dissolve the sealant 140 first slows down the flow-through ofthe liquid 144 through the pores 138. By not using the sealant 140, theliquid 144 of the droplet 126 would immediately start to flow throughthe pores 138 and because the droplet 126 is thickest at the center andthinner at its periphery more particles 142 tend to be located in themiddle of the droplet. This often results in an uneven distribution ofthe particles onto the filter membrane and the outer edge of the footprint of the particles sample is not clear. It should be noted moreparticles are not always located in the middle of the droplet becausesome specimen may have a tendency to concentrate towards the air-waterinterface. It is generally difficult to exactly foresee how differentsamples behave and distribute.

Since the entire footprint 136 of the sample droplet 126 is used tocalculate the particle concentration of particles 142, the droplet 126should not touch the inner edge of the filter holder of filter membrane116. Thus, it is important that only a defined part of the filtermembrane 116 is covered with the sample droplet 126. This is to makesure that all the particles 142 in the droplet 126 are enumerated orcounted. Also, the position of sample droplet 126 should be aligned withcavity 129 and channel 128 defined inside stub 120. Without pretreatmentof the filter membrane 116 with sealant 140, the surrounding filterpores, i.e. pores 138 a-138 d and 138 i-138 j in FIG. 6, remain open andair flows around the droplet 126 and through the filter membrane 116 sothat the sample droplet 126 does not absorb and becomes filtered fastenough to get a good sample distribution of the particles 142. In otherwords, the use of the sealant 140 has the advantage of creating a moredistinct outer periphery 137 of the footprint 136 of droplet 126 whenliquid starts to dissolve sealant 140 that is deposed below droplet 126.Without the use of sealant 140 there is not enough time for theparticles 142 to be evenly distributed inside droplet 126 since theliquid 144 immediately starts to flow through the pores 138 withoutgiving the particles 142 time to settle and be evenly distributed insidedroplet 126. One very important feature of sealant 140 is thus to createa vacuum condition so that a defined specimen footprint is formed. Moreparticularly, without the treatment of the sealant 140 according to thepresent invention, the droplet 126 undesirably dries through diffusionand evaporation. This results in a highly uneven particle distributiondue to the drying effects. It was surprisingly discovered that theundesirable evaporation may cause osmotic effects potentially causingparticle disruption and crystal formation due to increased saltconcentration in the remaining droplet. Additionally, this obscures theparticle detection and enumeration caused by broken particles andparticles that are hidden by salt precipitates. In the presentinvention, when applying the sample droplet 126 onto the sealed filtermembrane 116, the liquid 144 in the sample droplet 126 slowly dissolvesthe sealant 140 to open the pores 138 e-138 h disposed underneath thedroplet 126. Consequently, the liquid 144 is rapidly drawn through thepores 138 e-138 h of filter membrane 116 by the vacuum, resulting in agood sample distribution of particles 142 on the top surface of filtermembrane 116.

7. Before applying the sample droplet 126 onto the filter membrane 116,the vacuum device 102 is activated and the pressure in the vacuumchamber 104 is lowered to create suction on the filter membrane 116. Thevacuum in the vacuum chamber 104 ensures that the liquid 144 of droplet126 is absorbed evenly on the filter membrane 116. The combination ofthe usage of the sealant and the vacuum results in an even distributionof particles 142 across the footprint 136 on the filter membrane 116.

8. A suitable volume (typically 5 μl) of the sample droplet 126 isapplied on the porous and sealed filter membrane 116. As indicatedabove, it is important that the diameters of the particles 142 aregreater than the diameter of pores 138 of filter membrane 116 and thatthe droplet 126 does not touch the edges of the filter mount. A highervolume than 5 μl can be applied by using an injection system whereeither multiple drops or larger volumes are applied on the same positionon the filter membrane 116. In general, the use of larger volumesminimizes the sampling error and allows the analysis of lessconcentrated samples.

9. The sample droplet 126 is absorbed on the filter membrane 116 fortypically 60 seconds under vacuum pressure provided by vacuum chamber104. The exact pressure values may have to be adjusted partly dependingon pore size, sample type, volume, purity and viscosity.

10. After absorption, the filter membrane 116 may be detached from thefilter assembly 112 mounted onto the SEM alumina stub 120 (typically byusing an adhesive and conductive carbon tape 122).

11. The filter membrane 116, with bound particles 142 placed thereon,may then be sputter coated by for example a thin film of carbon(typically 20 nm thick) using a carbon evaporator at a suitable chamberpressure typically 1×10⁻⁵ mbar. The sputter coating improves theconductivity of the filter membrane 116; increases the signal to noiseratio of the filter membrane 116 and reduces the electron beam damageand charging effects. This technique is often necessary to use in orderto image a filter material using a SEM. It may be unconventional to usecarbon coating but it provides higher resolution SEM imaging compared tothe larger grain size of metal sputtering.

12. The filter membrane 116 may be transferred to the SEM and the signalfrom scattered primary electrons (using an in-lens detector) orsecondary electrons (such as by using a SE2 detector) is recorded bothat low to cover the entire footprint and high magnification (typically10 000 to 30 000) for enumeration. If a reference standard with adifferent secondary electron signature is used (albeit not necessary todetermine the particle concentration) the particles of interest can bedistinguished from the reference particles by combining intensityinformation from different detectors (such as in-lens and SE2detectors).

13. The low magnification images 136 (see FIG. 4B) are used to definethe size of the footprint of the droplet and the overall specimendistribution while the high magnification images are used to determinethe particle enumeration.

14. The high magnification images, such as image 134, are acquiredacross the sample footprint starting from the edge 137, through thecenter 139 and to the opposite edge of the droplet in order to minimizeany effect of differences in particle distribution across the footprintof the droplet.

15. From the low magnification images, such as image 136, the area ofthe sample footprint (A_(total)) is calculated by tracing the edge ofthe footprint. The encircled pixels are counted and the number countedis multiplied with the pixel size.

16. From the high magnification images, the particles 142 are detectedand counted. This procedure can be performed through manual marking orautomated marking by using suitable software such as Vironova'sproprietary software Analyzer or any other appropriate image analysissoftware. The average number of particles per area unit (n/A_(FOV)) iscalculated from the image dataset.

17. The number of particles per mL in the particle sample is,preferably, calculated by using the following formula:

$C = {A_{total} \times \frac{n}{A_{FOV}} \times {df} \times \frac{1000\mspace{14mu} {µl}}{V\mspace{14mu} {µl}}}$

Where C is the concentration of particles, df is the dilution factor andV is the applied volume of sample. It may also be possible to use aformula that takes into account that the particle distribution may varyfrom the periphery of the particle sample as the sample is scannedtowards the center thereof.

In summary, the particle quantitative scanning electron microscopy(pqSEM) technique of the present invention is a high-precision directparticle detection and enumeration technique. An important feature ofthe present invention is that the direct detection does not depend onthe affinity between a probe and the specimen which many existingconventional techniques do. All parameters, such as the dilution factor,the applied volume, the footprint of the droplet can be controlled andthe number of particles per area unit can be directly measured whileminimizing the error from approximations and assumptions. Moreover, theresolving power of the pqSEM permits detection of individual sub-visibleparticles within clusters and two populations of particles of differentsizes or other morphological features can be enumerated from the samesample. The particles and the footprint from the high-contrast imagesgenerated by the pqSEM technique of the present invention can readily bedetected by using automated image analysis. This provides the means forrapidly collecting large datasets and producing robust statisticalresults.

While the present invention has been described in accordance withpreferred compositions and embodiments, it is to be understood thatcertain substitutions and alterations may be made thereto withoutdeparting from the spirit and scope of the following claims.

We claim:
 1. A method for quantification of sub-visible particles,comprising: providing a filter membrane having a plurality of poresdefined therethrough; sealing the pores with a sealant; applying asample droplet, containing a liquid and sub-visible particles, onto thefilter membrane; the liquid dissolving the sealant in pores disposedbelow the sample droplet; the liquid flowing through the pores in whichthe sealant has been dissolved and the sub-visible particles remainingon top of the filter membrane; the sub-visible particles beingenumerated in an electron microscope; and wherein the method furthercomprises the step of pre-mounting a filter assembly containing thefilter membrane onto a support.
 2. The method of claim 1 wherein themethod further comprises the step of placing a mounting tape on a SEMsupport.
 3. The method of claim 1 wherein the method further comprisesthe step of providing the SEM support having an elongate channel definedtherein, using an injector containing the sample droplet, and aligningthe injector on top of an elongate channel prior to depositing thesample droplet on the filter membrane.
 4. The method of claim 1 whereinthe method further comprises the step of connecting the SEM support to avacuum chamber connected to a vacuum source and subjecting the filtermembrane to a suction force via the elongate channel.
 5. The method ofclaim 1 wherein the method further comprises the step of the liquid onlydissolving the sealant in the pores disposed immediately below sampledroplet while adjacent pores remain sealed with the sealant.
 6. Themethod of claim 1 wherein the method further comprises the step of thesub-visible particles forming a footprint on the filter membrane andscanning the sub-particles from an outside periphery of the footprinttowards a center of the footprint.
 7. The method of claim 1 wherein themethod further comprises the step of using a diluent of the liquid todissolve the sealant in the pores located directly below the sampledroplet.
 8. The method of claim 1 wherein the method further comprisesthe step of using glycine as the sealant.
 9. The method of claim 1wherein the method further comprises placing the filter membrane beingin operational engagement with a vacuum chamber.
 10. The method of claim1 wherein the method further comprises applying the sample droplet ontothe filter membrane without the sample droplet touching any outside edgeof the filter membrane.